DESCRIPTION (provided by principal investigator): Glaucoma remains a major blinding disease affecting over 66 million persons worldwide. This project has focused on the molecular mechanism(s) by which laser trabeculoplasty (LTP) ameliorates the elevated intraocular pressure (IOP) associated with most of primary open-angle glaucoma. Our studies indicate that extracellular matrix (ECM) turnover within the trabecular meshwork (TM), triggered by the cytokines TNF and IL-1 is a key component of the efficacy of LTP. Building on our recent results with this system, our focus now is to identify the molecular component(s) that comprise the aqueous humor outflow resistance and regulate IOP. Our working hypothesis is that the glycosaminoglycans (GAGs) on one or more specific proteoglycans within the juxtacanalicular region of the trabecular meshwork (TM) and Schlemm's canal inner wall endothelium form a critical component of the aqueous humor outflow resistance. Aim 1 will be to identify which specific GAGs are changed coincident with several manipulations that change the outflow resistance in perfused anterior segment organ culture. These manipulations include GAG degrading enzymes, GAG biosynthesis inhibitors, TNF1, IL-11, TGF22 and increased IOP. Segmental differences in outflow rates within eyes will also be compared. Subtle differences between human and porcine outflow responses will be used to further refine the identification. Standard light and confocal microscopy using GAG stains, antibodies, binding proteins and lectins with enzymatic degradation will be used. Aim 2 will be to identify the specific proteoglycan(s) that are the core proteins for the GAGs that provide the resistance. The same manipulations will be used with a combination of immunohistochemistry and biochemical methods to identify the core protein. Aim 3 will be to verify the identification of the resistance-causing GAG/proteoglycan by alternative methods. The effects on outflow of silencing the key GAG biosynthetic enzyme and the proteoglycan core with shRNA and of perfusing expressed proteoglycan binding domains will be determined. Aim 4 will be to use these binding domains to localize and identify binding partners and unravel the molecular and structural organization of the outflow resistance. Using these divergent approaches, we plan to determine, for the first time, the specific molecular identity of the GAG/proteoglycan(s) responsible for the aqueous humor outflow resistance and begin elucidating the structural organization of the resistance. Identifying the key molecular constituent of the outflow resistance should facilitate development of improved therapies for primary open-angle glaucoma. PUBLIC HEALTH RELEVANCE. Glaucoma remains the second leading cause of permanent vision loss in developed nations. The cause(s) of this disease are not well understood and therapies are directed at treating the symptoms. These studies are focused at understanding the molecular nature of the aqueous humor outflow pathway, which is normally responsible for avoiding glaucoma and which becomes obstructed to produce glaucoma.